Diagnosis of whiplash associated disorders (wad) by using pet with d-[methyl-11c]-deprenyl (dde)

ABSTRACT

Positron Emission Tomography (PET) tracers such as D-[methyl- 11 C]-Deprenyl (DDE) and [ 11 C]-GR205171 (GLD), methods for and methods of preparing biological mechanisms that identify treatment targets in connection with Whiplash-Associated Disorder (WAD) are provided. Associated kits for the evaluation of the biological mechanisms are also provided.

FIELD OF THE INVENTION

The present invention relates to the development of a Positron EmissionTurnover (PET)-tracer that could be used to diagnose awhiplash-associated disorder (WAD). The present invention furtherrelates to methods for the diagnostic use of a PET tracer that can beused for WAD, The present invention further relates to the studies ofunderlying biological mechanisms that could contribute to identifyingnew diagnosis and treatment targets for WADs.

BACKGROUND OF THE INVENTION

The commonly used term Whiplash is defined as anacceleration-deceleration mechanism of energy transfer to the neck whichmay result from rear-end or side impact, predominately in motor vehicleaccidents and from other mishaps. The energy transfer may result in bonyor soft tissue injuries (whiplash injury) which may in turn lead to awide variety of clinical manifestations such as whiplash associateddisorders. (Spitzer W O, Skovron M L, Salmi L R et al., Scientificmonograph of the Quebec Task Force on WAD: redefining whiplash and itsmanagement”, Spine, 1995).

Whiplash injuries are common; the incidence has been estimated to beapproximately 4 per 1000 people (Barnsley L, Lord S., Bogduk N.,“Whiplash injury”, Pain, 1994). Although many people involved in motorvehicle accidents recover quickly, between 4% and 42% of patients withaccident-related neck injuries report symptoms several years later.(Lord S M, Barnsley L., Wallis B J, Bogduk N., “Chronic cervicalzygapophysial joint pain after whiplash: a placebo-controlled prevalencestudy”, Spine, 1996).

Chronic pain syndromes which include whiplash-associated disorders (WAD)are prevalent, cause significant individual suffering, and are highsocietal costs. In contrast to other injuries due to traffic accidents,neck injuries have increased. For example, neck injuries associated withat least 10% disability have increased from roughly 30 to roughly 60%during the two latest decades.

Like chronic pain syndromes, WAD causes significant individual sufferingas well as high societal costs. For example, over 29 billion dollars peryear is spent on whiplash injuries and litigation in the United States.Roughly ⅓rd of all WAD patients develop chronic problems. (Lord SM,Barnsley L., Wallis B J, Bogduk N., “Chronic cervical zygapophysialjoint pain after whiplash: a placebo-controlled prevalence study”,Spine, 1996).

A continuous search is under way for new treatment modalities of WAD,but at present, much work is focused on the early identification ofpatients with WAD. Although generally accepted diagnostic criteria forWAD as well as outcome measures exist, prognostic markers are lacking.Present methods do not allow important subgroups of patients to beclearly distinguished. The need for more precise methods to determineseverity and prognosis as well as a treatment response is urgent.Conventional radiology only gives information about already establishedinjuries. Magnetic Resonance Imaging tomography (MRI), computertomography (CT) and radioisotope methods have shown potentials ofvisualizing signs of biological processes, but more work is neededbefore they can be used as an objective and quantitative assessment ofWAD and different therapeutic strategies.

Positron emission tomography (PET) imaging is not currently used in thediagnosis of WAD, although PET has been used to demonstrate high glucosemetabolism in joints of rheumatoid arthritis patients. PET has also beenused in conjunction with the tracer D-[methyl-¹¹C]-deprenyl (also knownas DDE or ¹¹C-D-deprenyl) in diagnosing and treating patients witharthritis. (Danfors T., Bergstrom, M. et al., “Positron EmissionTomography with ¹¹C-D-deprenyl in Patients with Rheumatoid Arthritis”,Scan J. Rheumatol, 1997).

Furthermore, DDE has been used as a negative control for thedemonstration of selective MAO-B-binding by ¹¹C-L-deprenyl.¹¹C-L-deprenyl is an enantiomer of DDE. An enantiomer exists when achemical structure and its mirror image are not superposable. In a rangeof pituitary adenomas, ¹¹C-L-deprenyl showed significant retention inboth a tumourous and normal brain, whereas in most of the tumours, DDEshowed a rapid washout from both tumour and normal brain. However, insome tumours, DDE was retained in the tumour but not in the normalbrain. The washout suggested that DDE had negligible binding to MAO-B,but the retention in some tumours suggested an additional mechanism ofretention ofDDE. (Danfors T., Bergstrom, M. et al., “Positron EmissionTomography with ¹¹C-D-deprenyl in Patients with Rheumatoid Arthritis”,Scan J. Rheumatol, 1997). MAO-B is monoamine oxidase B which isidentified as a member of the family of the imidazoline bindingproteins.

Moreover, there has yet to be found a PET-tracer that could be used forWAD. Accordingly, there has been a long felt need for the development ofa PET-tracer that could be used to diagnose WAD as well as studyunderlying biological mechanisms that retain a PET-tracer which couldcontribute to identifying new diagnosis and treatment targets for WAD.

Discussion or citation of a reference herein shall not be construed asan admission that such reference is prior art to the present invention.

SUMMARY OF THE INVENTION

In view of the long felt need for diagnosing whiplash-associateddisorder (WAD), the present invention relates to both the development ofa PET-tracer that could be used to diagnose WAD and the study ofunderlying biological mechanisms that could contribute to identifyingnew diagnosis and treatment targets for WADs.

In one embodiment, a Positron Emission Tomograhpy (PET) tracer fordiagnosing whiplash-associated disorder is disclosed wherein the PETtracer is D-[methyl-¹¹C]-Deprenyl DDE). The PET tracer could also be[¹¹C]-GR205171 (GLD).

In a further embodiment, a biological mechanism that is a specificmolecular structure or enzyme which is expressed in the inflammed jointtissue, wherein the biological mechanism could contribute to identifyingtreatment targets according to WADs is also disclosed.

In another embodiment, a method for the preparation of an enzyme ormolecular structure according to the steps of using capillaryelectrophoresis to aid in separating out proteins such as MAO-Bproteins; and then detecting the radioacitivity by ¹¹C-labelledprecursors to guide a skilled artisan as to which MAO-B protein wouldbind to either DDE and GLD is also presented.

The present invention further provides for a kit in the preparation of amolecular structure or enzyme according to the steps of using capillaryelectrophoresis to aid in separating out proteins such as MAO-Bproteins; and then detecting the radioacitivity by ¹¹C-labelledprecursors to guide which MAO-B protein would bind to either DDE andGLD.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 shows a PET-imaging picture that depicts an increased DDE uptakein one of the WAD patients.

DETAILED DESCRIPTION OF THE INVENTION

The present invention relates to examining patients with Whiplashassociated-syndrome (WAD) by investigating the presence of localchanges, caused by local micro-lesions and producing an increased¹¹C-D-deprenyl (DDE) uptake revealed through Positron EmissionTomography (PET) that is impossible to be revealed by computertomography (CT) or Magnetic Resonance Imaging tomography (MRI).

PET imaging is a tomographic nuclear imaging technique that usesradioactive tracer molecules that emit positrons. When a positron meetsan electron, they both are annihilated and the result is a release ofenergy in the form of gamma rays, which are detected by the PET scanner.By employing natural substances that are used by the body as tracermolecules, PET does not only provide information about structures in thebody but also information about the physiological function of the bodyor certain areas therein. Furthermore, the choice of a tracer moleculedepends on what is being scanned. Generally, a tracer is chosen thatwill accumulate in the area of interest, or be selectively taken up by acertain type of tissue, e.g. cancer cells. Scanning consists of either adynamic series or a static image obtained after an interval during whichthe radioactive tracer molecule enters the biochemical process ofinterest. The scanner detects the spatial and temporal distribution ofthe tracer molecule. PET also is a quantitative imaging method allowingthe measurement of regional concentrations of the radioactive tracermolecule. Commonly used radionuclides in PET tracers are ¹¹C, ¹⁸F, ¹⁵O¹³N or ⁷⁶Br.

Furthermore, tracers labeled with short-lived positron emittingradionuclides (e.g. ¹¹C, t_(1/2)=20.3 min) are frequently used invarious non-invasive in vivo studies in combination with PET. Because ofthe radioactivity, the short half-lives and the submicromolar amounts ofthe labeled substances, extraordinary synthetic procedures are requiredfor the production of these tracers. An important part of theelaboration of these procedures is the development and handling of new¹¹C-labelled precursors. This is important not only for labeling newtypes of compounds, but also for increasing the possibility of labelinga given compound in different positions.

When compounds are labeled with ¹¹C, it is usually important to maximizespecific radioactivity. In order to achieve this, the isotopic dilutionand the synthesis time must be minimized. Isotopic dilution fromatmospheric carbon dioxide may be substantial when [¹¹C]carbon dioxideis used in a labeling reaction. Due to the low reactivity andatmospheric concentration of carbon monoxide (0.1 ppm vs. 3.4×10⁴ ppmfor CO₂), this problem is reduced with reactions using [¹¹C]carbonmonoxide.

There are several advantages in using the PET technique in the diagnosisof WAD. One advantage is that the PET technique offers a potential forrecording various functional and biochemical characteristics in theaffected joints. Another advantage is that the PET technique has apotential to supply objective and quantitative estimates of diseaseintensity rather than secondary structural information.

One embodiment of the invention is to provide a Positron EmissionTomograhpy (PET) tracer for diagnosing whiplash-associated disorderwherein a PET tracer is D-[methyl-11-C]-Deprenyl (DDE). The PET tracercan also be [11C]-GR205171 (GLD).

Another embodiment of the present invention includes either DDE or GLDor in combination thereof can be used for joint diseases comprisingarthritis, rheumatoid arthritis, gout, osteoarthritis, ankylosis,bursitis, temporomandibular joint disorders, synovial chrondromatosis,hemarthrosis, acquired joint deformalities, metatarsalgia, arthralgia,arthrogryposis, joint instability, synovitis, neurogenic arthropathy,hallux rigidus, hydrathrosis, joint loose bodies, and similar diseasesthereof.

In a further embodiment, a biological mechanism that is a specificmolecular structure or enzyme which is expressed in the inflammed jointtissue of a patient, wherein the biological mechanism identifiestreatment targets according to WADs is also disclosed. A patient usedherein is a human being or any kind of animal thereof.

In yet another embodiment, a method for the preparation of a molecularstructure or an enzyme according to the steps:

-   -   i) using capillary electrophoresis to aid in separating out        MAO-B proteins; and then    -   ii) detecting the radioactivity by ¹¹C-labelled precursors to        guide a skilled artisan as to which MAO-B protein would bind to        either or both DDE and GLD is also presented. A skilled artisan        used throughout this application is defined as a person who is        of skill in this particular field.

The present invention further provides a kit for the preparation of amolecular structure or an enzyme according to the steps:

-   -   i) using capillary electrophoresis to aid in separating out        MAO-B proteins; and then    -   ii) detecting the radioactivity by ¹¹C-labelled precursors to        guide a skilled artisan as to which MAO-B protein would bind to        either or both DDE and GLD is also presented.

Yet another embodiment of the present invention provides for adiagnostic use of a PET tracer comprising D-[methyl-11-C]-Deprenyl(“DDE”) or [11C]-GR205171 (“GLD”) for determining WAD.

Still a further embodiment encompasses a method of use for generating abiological mechanism that is a specific molecular structure which isexpressed in the inflammed joint tissue of a patient, wherein thebiological mechanism identifies treatment targets according to WADs.

While still another embodiment entails a method of use for generating abiological mechanism that is an enzyme which is expressed in theinflammed joint tissue of a patient, wherein the biological mechanismidentifies treatment targets according to WADs.

Another embodiment of the present invention entails a method of use forpreparing a molecular structure according to the steps:

-   -   i) using capillary electrophoresis to aid in separating out        MAO-B proteins; and then ii) detecting the radioactivity by        ¹¹C-labelled precursors, whereby guiding the MAO-B proteins to        either bind to DDE, GLD or both.

A further embodiment of the present invention encompasses a method ofuse for preparing an enzyme according to the steps:

-   -   i) using capillary electrophoresis to aid in separating out        MAO-B proteins; and then    -   ii) detecting the radioactivity by ¹¹C-labelled precursors,        whereby guiding the MAO-B proteins to either bind to DDE, GLD or        both.

EXAMPLES

The invention is further described in the following examples which arein no way intended to limit the scope of the invention.

Uppsala Imanet has developed two tracers which areD-[methyl-¹¹C]-deprenyl (DDE) and [11C]-GR205171 (GLD) that are to beused in combination with a Positron Emmison Tomography (PET) scanner,for clinical investigations indicating an inflammatory process such asWhiplash Associated-Disorder (WAD).

In one experiment, eight patients have been examined and diagnosed withWAD through the use of DDE and PET. In four patients DDE appeared tobind to soft tissue in the neck vertebra. Clinically, those patientswith a higher uptake of DDE in the affected joint areas seem to reporthigh pain ratings while being in the scanner.

Furthermore, when a patient was administered intra-articular injectionsof gluccorticoids a significant diminishing of the uptake of DDE in theaffected area, such as but not limited to the synovial (or diarthrodial)joint or similar joints, was observed 6-14 days after treatment. Theeffects are observed with all modes of evaluation: After treatment, theintitial high uptake of DDE in the affected joint is significantlydiminished, indicating a pronounced effect on perfusion.

In a similar experiment, sixteen patients were investigated using a highresolution PET scanner. It was observed that about 50% of the patientshad an increased uptake of DDE in the neck and shoulder areas of thepatients. As an example, FIG. 1 shows a PET-imaging picture that depictsan increased DDE uptake in one of the WAD patients.

In a third experiment eight healthy subjects were investigated using aPET-CT (Computerized Tomography) scanner. PET/CT combines the strengthsof two well-established imaging modalities, PET for function and CT foranatomy, into a single imaging device. The subjects were free from painin the neck and shoulder regions during at least two months and they hadnot been involved in accidents which could have caused a neck injury.None of the subjects revealed an increased uptake of DDE in the scannedneck and shoulder regions. This indicates that the increased uptake inpatients (experiments 1 and 2) are related to their clinical symptoms.

Furthermore, on-going analysis are being performed throughsemi-quantitative analysis by comparing the neck and shoulder areas wihan increased DDE uptake to an area of the body without any DDE uptake.Additionally, a control material is planned to compare WAD-patients andhealthy volunteers.

In search for a mechanism of retaining DDE, studies regarding inflamedjoints such as synovial (or diarthrodial) were adminstered. The resultsuncovered a very pronounced uptake and retention on swollen synovialtissue, correlating well with clinical and others signs of activeinflammation. In a separate study, an investigation pertaining to theradioactivity concentration in synovial fluid of patients withrheumatoid arthritis given the tracer DDE was adminstered. The synovialfluid had low radioactivity whereas the inflamed tissue as seen in thePET images had very high activity. This activity was reduced by 50% theday after intra-articular administration of glucocorticoid.

There are various hypothesis as to what biological mechanisms are behindthe high uptake of DDE in inflammatory joint tissues of humans. Onehypothesis is binding DDE to a specific molecular structure or enzymewhich is expressed in the inflamed area. To validate the hypothesis afrozen section autoradiography was performed on various tissues usingDDE and thereby demonstrating a much higher binding in inflamed jointsthan any other tissues. In further determining the character of thebinding between DDE and inflammatory joint tissues, it is anticipatedthat capillary electrophoresis would aid in separating out proteins andthe detection of the radioactivity would guide one to know how many andpotentially which protein is binding to the tracer.

In addition, in order to exclude the possibility that the DDE uptake isgoverned by binding to a monoamine oxidase B (MAO-B), a reexamination ofone patient after adminstration of Eldepryl demonstrated to besufficient for blocking of an enzyme. In this patient no blocking effectcould be seen, only a slightly higher uptake of DDE in the inflamedjoint area. In the human brain and pituitary adenomas it has been shownthat ¹¹C-L-deprenyl has a higher binding, whereas DDE is rapidly washedout except in subgroups of non-secreting pituitary adenomas where DDEshows high binding.

Furthermore, it has been hypothesized that the pharmacological challengeof the binding between DDE and inflammatory joint tissues in patients iswhen inflamed synovial tissue is incubated with DDE, with or without arange of MAO-B binding proteins or similar compounds which couldpotentially inhibit the binding. The inhibitors that could be used topotentially inhibit the binding between DDE and inflammatory jointtissue are inhibitors of VAP-1 and other cell surface amino oxidases.Additionally, other molecular entities are searched for which wouldserve the same task of binding to the identified molecular target.

In addtion, in a small pilot series of experiments in patients withbacterial abscesses, no visible increase of DDE or GLD uptake was found.

Furthermore, in vitro binding experiments demonstrated very high DDE andGLD tracer binding in inflammatory tissues removed from patients withrheumatoid arthritis. The in vitro binding experiments were performed onthin slices of tissue of about 20 micrometers in rats and in humans. Thethin slices of tissues were adhered to gelatinized slide glasses. Thetissue adhered to the glass was then incubated in a buffer such asTRIS-HCl containing DDE or GLD at about 2 nM concentration for about 40minutes at room temperature. The experimental conditions were varied andthe results were not significantly affected. After incubation the slideglasses were washed about 3 times in a buffer, dried, and exposed onphosphor imaging plates for a minimum of 60 minutes.

In the in vitro binding experiments, local uptake was present in allcases in the parotis and submandibularis glandulae. Differences betweenpatients were found in areas related to the insertion of muscles to theoccipital bone, vertebrae and clavicula. In these regions, four of theexamined patients showed a visually enhanced uptake in comparison withthe rest. In some of these patients an increased uptake was observed inde proximal and distal parts of the muscle sternocleidomastoideus.

In order to make inter-individual comparisons, it was decided tonormalize the uptake in the above-mentioned areas with the uptake of theinferior part of the cerebellum. In the area of insertion of m. rectuscapitis posterior major we found bilaterally in 3 patients an increaseof 57%-127% in the uptake of DDE compared with the cerebellum. Thefourth patient did not show a clear increase, but there was a 40%difference between right and left side of the cerebellum.

At the level of the cervical vertebrae 2 and 3 (insertion in m.semispinalis capitis) it was found in one case an increase ofapproximately 70% uptake of DDE and GLD. The other two patients showedlower uptake of these two PET tracers but differences in the patientswere found between both sides of the cervical vertebrae in the order of16-30%. High uptake of DDE and GLD at the level of insertion in theclavicula 70-120% was found in some of these four patients.

Specific Embodiments, Citation of References

The present invention is not to be limited in scope by specificembodiments described herein. Indeed, various modifications of theinventions in addition to those described herein will become apparent tothose skilled in the art from the foregoing description and accompanyingfigures. Such modifications are intended to fall within the scope of theclaims.

Various publications and patent applications are cited herein, thedisclosures of which are incorporated by reference in their entireties.

1. A method for diagnosing whiplash-associated disorder (WAD) in asubject comprising: (a) administering an effective amount ofD-[methyl-¹¹C]-deprenyl (DDE) to a subject; and (b) performing a PETscan on said subject.
 2. (canceled)
 3. A PET tracer according to claim1, wherein the PET tracer comprises [11C]-GR205171 (GLD). 4-15.(canceled)
 16. The method of claim 1, wherein said PET scan comprises aPET-CT scan.
 17. The method of claim 1, wherein said PET scan isperformed on the neck region of said subject.
 18. The method of claim17, wherein said PET scan shows an increase in DDE uptake in the neckregion of said subject.
 19. The method of claim 1, wherein said PET scanis performed on the shoulder region of said subject.
 20. The method ofclaim 19, wherein said PET scan shows an increase in DDE uptake in theshoulder region of said subject.
 21. A method for PET imaging a subjectbelieved to be suffering from whiplash-associated disorder (WAD), themethod comprising: (a) administering an effective amount ofD-[methyl-¹¹C]-deprenyl (DDE) to a subject; and (b) performing a PETscan on said subject.
 22. The method of claim 21, wherein said PET scancomprises a PET-CT scan.
 23. The method of claim 21, wherein said PETscan is performed on the neck region of said subject.
 24. The method ofclaim 23, wherein said PET scan shows an increase in DDE uptake in theneck region of said subject.
 25. The method of claim 21, wherein saidPET scan is performed on the shoulder region of said subject.
 26. Themethod of claim 25, wherein said PET scan shows an increase in DDEuptake in the shoulder region of said subject.
 27. A method fordiagnosing whiplash-associated disorder (WAD) in a subject comprising:(a) administering an effective amount of [11C]-GR205171 (GLD) to asubject; and (b) performing a PET scan on said subject.